Apparatus for investigating the condition of a bacteria containing suspension through frequency profile of electrical admittance

ABSTRACT

A method of investigating the condition of biological particles, in particular the fraction of viable cells in a bacteria population involves applying a voltage and monitoring the voltage induced by magnetic induction in a detector coil. The applied voltage is of a variable frequency alternating voltage and is preferably induced in the medium through a drive coil.

This is a divisional application of our earlier application Ser.No.829,644 filed on Jan. 31, 1986 filed as PCT GB85/00150 on Apr. 3,1985, published as WO85/04481 on Oct. 10, 1985, that issued as U.S. Pat.No. 4,810,963 on Mar.7, 1989 .

FIELD OF THE INVENTION

This invention relates to a method and apparatus for investigating thecondition of biological particles, such as bacteria, in a medium. In oneexample the invention is concerned with establishing what fraction ofthe bacteria are viable.

BACKGROUND OF THE PRIOR ART

Work has previously been carried out in investigating the electricalimpedance of suspensions of certain bacteria. It has been establishedthat the impedance consists primarily of resistive and capacitativecomponents, though the measured conductivity and relative permittivityare not constant but functions of frequency. In a plot of relativepermittivity against frequency, it is possible to identify alpha andbeta dispersions which are believe to relate to the respectivecontributions of different membrance layers in the bacterium. Eachmembrane layer is believed to function, as a resistance/capacitancenetwork with the relative contribution of the various layers changingwith frequency. The alpha dispersion has been associated with the outermembrane layers and the beta dispersion with the innermost layers.

The traditional approach to the measurement of impedance of bacteriumsuspensions involves manual bridge techniques. These are time consumingand require very careful attention to balancing and calibration. Theprocedures do not lend themselves to use in a bioreactor on a commercialscale where real time, continuous monitoring is required.

A number of commercially available impedance analysers have beensuggested for use in investigating dieletric dispersion in suspensions.The difficulty here is that the impedance analysers are constructed andorganized to operate on networks of unknown but constant resistance,capacitance and inductance. Very careful further analysis is required,bearing in mind that the conductivity and relative permittivity of thesuspension are known to be functions of frequency, before the output ofa commercially available impedance analyser can provide usefulinformation in this context.

SUMMARY OF THE INVENTION

It is an object of one aspect of this invention to provide improvedmethod and apparatus for investigating the concentration of biologicalparticles which is capable of distinguishing, for example, betweenviable and non-viable bacteria.

Accordingly, the present invention, in one aspect, consists of a methodof investigating the condition of biological particles in a medium,comprising the steps of applying an alternating voltage of varyingfrequency across a body of medium; monitoring the voltage induced in adetector coil adjacent the medium and deriving a frequency profile ofcomposite medium and particle admittance.

In another aspect, the present invention consists in apparatus forinvestigating the condition of biological particles in a medium,comprising a chamber filled in use with the medium; electrode means forapplying a voltage across medium in the chamber; voltage generator meansfor generating an alternating voltage of selected frequency at saidelectrode means; coil means so disposed that the voltage induced thereinis indicative of the magnetic field associated with current flow in themedium and processor means for compiling a profile of measured voltagein the coil means against frequency of voltage applied to the electrodemeans.

Advantageously, the processor comprises storage means for storing theprofiles.

Suitably the chamber comprises a conduit through which medium may flowcontinuously and, preferably, the conduit is cylindrical and the coilmeans comprises a helical coil having its axis orthogonal to the axis ofthe cylinder.

In another form, the present invention consists in apparatus forinvestigating the condition of biological particles in a medium,comprising a chamber filled in use with the medium; drive coil means forinducing a voltage in the medium; voltage generator means for applyingan alternating voltage of variable frequency to said drive coil means,detector coil means so disposed that the voltage induced therein isindicative of the magnetic field associated with current flow in themedium and processor means for compiling a profile of measured voltagein the detector coil means against frequency of voltage applied to thedrive coil means.

Advantageously, said voltage generated means is adapted to vary themagnitude of the generated alternating voltage so as to maintain aconstant magnitude current in the drive coil at varying frequencies.

By deriving an impedance/frequency profile for the same suspension atdifferent times, it will be possible through comparison to detect ashift in weighting of the alpha and beta dispersions identifiable withthe change in the proportion of viable bacteria. In the example of abacterium it is necessary to distinguish between viable bacterium and"ghosts" comprising ruptured cell walls from which the cytoplasm hasbeen lost. It is believed that the "ghost" will contribute to the alphadispersion, which is associated with the outer cell layers but will makeno contribution to the beta dispersion. A relative increase in theweighting of the alpha dispersion can thus be interpreted as a decreasein the number of viable cells.

In another aspect, the present invention consists in a method ofinvestigating the concentration of biological particles in a mediumcomprising the steps of amplifying a voltage induced by alternatingcurrent flow through a body of the medium; applying said voltage to themedium to excite resonance and determining the frequency of resonance.

In yet a further aspect, the present invention consists in apparatus forinvestigating the concentration of biological particles in a medium,comprising a chamber filled in use with the medium; electrode means forapplying a voltage across medium in the chamber; coil means so disposedthat the voltage induced therein is indicative of the magnetic fieldassociated with current flow through the medium; amplifier means forpresenting said induced voltage to the electrode means in a manner toexcite resonance and means for measuring the resonant frequency.

BRIEF DESCRIPTION OF THE DRAWING

The invention will now be described by way of example with reference tothe accompanying drawings in which:

FIG. 1 illustrates somewhat diagrammatically a test cell for use in thisinvention;

FIG. 2 is a block diagram of apparatus according to this inventionincluding the test cell of FIG. 1;

FIGS. (3a), (b) and (c) are illustrative frequency plots;

FIG. 4 is a block diagram of alternative apparatus according to thisinvention;

FIG. 5 is a partly diagrammatic view of apparatus according to anotherembodiment of the invention;

FIG. 6 illustrates a variation of test cell for use in the presentinvention; and

FIGS. (7a) (b) and (c) are illustrative frequency plots; and

FIG. 8 is a diagram of yet further apparatus according to thisinvention.

DESCRIPTION OF THE PREFERRED EMBODIMENT

Referring to FIG. 1, a test cell according to this invention is showncomprising a silica or pyrex glass tube 10 having electrodes 12 and 14bonded to each end thereof. The electrodes are identical and comprise acup portion 16 which surrounds the end of the glass tube, the interiorsurface of the cup portion being formed with a rebate to ensure a smoothcomposite surface. The electrode has an inner conical surface 18 whichopens to an aperture 20 and serves as a sample fluid inlet/outlet. Anelectrode connector 22 of conventional form is provided on eachelectrode. Between the electrodes, the tube 10 carries on its outersurface a tightly wound helical coil 24. The coil 24 is arranged onsuitable formers (not shown) with the axis of the coil orthogonal to theaxis of the tube.

It will be understood that with a conductive medium in the glass tube,the application of an alternating voltage across electrodes 12 and 14will result in the passage of a current in the medium, this in turncreating an oscillating magnetic field inducing a voltage across thecoil, the turns of which cut the field.

With reference to FIG. 2, a system will now be described for derivinginformation from this induced voltage and its variation with frequency.

The test cell which is shown in FIG. 1 is connected with a mediumreservoir 30, a peristaltic pump 32 and a water bath 34 so as toestablish a uniform flow of a sample fluid through the cell at aconstant temperature. In an alternative arrangement, a sample of fluidmay be diverted continuously from a chamber or feed line of a bioreactorand passed through the temperature bath to the cell. A signal generator36 is connected with the electrodes 12 and 14 to apply across thoseelectrodes a sinusoidal voltage of fixed amplitude and of a selectedfrequency. Processor 38 is connected with the signal generator 36 tocause the signal generator to sweep through a frequency range. Theprocessor 38 enables preselection of the signal amplitude, the number offrequency sample points, the delay between samples, the number of repeatsamples of any one frequency and the frequency range of interest.

The alternating voltage induced in the coil 24 is passed to an amplifier40 having a high gain at low frequencies. The response of the amplifieris selected to compensate at low frequencies for the frequency variationof induced voltage according to Faraday's law. At higher frequencies,the induced voltage is sufficiently large not to require amplification.The output of the amplifier 40 is passed through analogue-to-digitalconverter 42 to the processor 38.

Through suitable sampling, the processor compiles a profile of inducedvoltage against frequency, including--say--300 data values in aparticular frequency band. An example of such a profile for an E Colisuspension is shown in FIG. (3a) . A qualitative comparison reveals thatthere has been a shift in the relative weights of the alpha and betadispersions which is indicative of a change in the proportion of viablebacteria.

In order to enable quantitative evaluation, a profile can be compiledfor medium from which bacteria have been separated. This may beachieved, for example, using the apparatus shown in FIG. 4 where theflow of sample may be taken either directly to the test cell or througha filter 50 arranged to separate bacterium or other biologicalparticles. The numerical profile generated for medium devoid of bacteriais stored in the processor and used as a baseline for evaluation ofsubsequent profiles. An example of a frequency plot for medium only isshown at FIG. (3b).

The result of subtracting the plot of FIG. (3b) (medium only) from theplot of FIG. (3a) (medium +E Coli) is shown in plot I of FIG. (3c). Theportion between the maximum at approximately 100kHz and the minimum atapproximately 350kHz can be interpreted as the alpha dispersion with thedifference in voltage terms between these two points being indicative ofthe strength of the alpha dispersion. As mentioned above, the alphadispersion is associated with the outer cell membranes. Plot IIcorresponds to a suspension in which a proportion of the bacteria havedied. It can be seen qualitatively that the strength of the alphadispersion has increased. For a quantitative analysis a comparison ofthe relative weightings of the alpha and beta dispersions is made, thebeta dispersion being found at higher frequencies--typically 50 MHz.

Referring now to FIG. 5, there is shown a further embodiment of thisinvention. A recirculating pathway for medium is established throughparallel paths 110a, 110b; a reservoir 112; a temperature controlledbath 114 and peristaltic pump 116. Parallel limb 110b carries on itsouter surface a coil 118 wound with its axis orthogonal to the pathwayand parallel to the plane of the paper. Under control from a processor120 a signal generator 122 applies an alternating voltage across thecoil 118 at a selected and variable frequency. The connection betweenthe signal generator and the coil includes a series precision resistor124 and a tap is taken from the resistor to enable current sensingwithin the signal generator.

It is important that, whatever the frequency, a constant drive currentshould flow in the coil 118 and there is therefore contained within thesignal generator 122 a comparator which produces an error signal if thesensed current (as represented by the voltage across the precisionresistor 124) departs from a preset current level. If the sensed currentdrops below the desired current, the amplitude of the voltage applied bythe signal generator is increased. To enable this condition of constantcurrent to be met over a useful range of frequencies, the signalgenerator includes a power amplifier.

The oscillating magnetic field created by the coil 118 will induce avoltage in the medium causing current to flow along the length of thepathway. This current will tend to circulate round parallel pathways110a and 110b. To detect this current, a detector coil 126 is disposedon the outside of limb 110a. This coil is similar to coil 118 but hasits axis orthogonal to the plane of the paper. The voltage induced inthe detector coil 126 is amplified, rectified and passed to data logger128. The processor 120 is further connected in conventional manner witha printer 130, a plotter 132, a VDU 134 and a keyboard 136.

In operation, the processor causes the frequency of the voltage appliedto the drive coil 118 to sweep through a desired frequency band. Asdescribed, it is ensured that the current in the drive coil is sensiblyconstant. Throughout the frequency sweep, the voltage in the detectorcoil 126 is sampled. In one example, a suspension of E Coli of volume14.5% in a nutrient medium was passed through the parallel limbs 110a,110b. The frequency of the applied voltage was swept from 500 Hz to 500kHz. A profile of detector coil voltage against frequency was producedand is shown at FIG. 7a. The frequency sweep was repeated with mediumfrom which bacteria had been removed by filtration and profile for themedium alone is shown at FIG. 7b. In this Figure, the dot dash linerepresents a calibration line achieved with an ohmic liquid in the flowpath. By subtracting the profiles of FIGS. (7a ) and (7b) , the profileof FIG. FIG. ) is produced which contains the desired informationconcerning the bacteria alone. In this profile of FIG. (7c ), the alphadispersion is clearly visible. With repeated measurements during thelife of a population of bacteria, changes in the weighting of the alphadispersion can be measured as described above.

In another modification a test cell suitable for use at very highfrequencies is shown in FIG. 6 where the glass tube of FIG. 1 has beenreplaced by a flexible PTFE tube 152 wound about a non-conductive former154. The coil 154 is open wound about the PTFE tube 152. In anotherrespect this modified test cell is used in a manner analogous to thatdescribed in FIG. 1.

In yet a further alternative arrangement according to the invention, thetest cell is incorporated into a closed oscillatory circuit such thatthe cell contents have the dominant role in determining the resonantfrequency of the system. This is achieved by using the flux linkagebetween the conductive cell contents and the feed coil as the positivefeedback path between the input and output of a high gain amplifier. Theamplifier response can be wideband or frequency controlled dependingupon the application; but in either case may be so designed as to havean amplitude dependent gain system thereby allowing the system tomonitor fluids where a reactive component is the primary variable or themeasured range of conductivities is too low to maintain oscillation.Referring to FIG. 8, the detector coil is connected to the highimpedance input of an amplifier 160, and, as required, the drive coil orelectrodes are supplied from the low impedance output of the amplifier.In practice, one end of each coil or one of the electrodes are connectedto ground. An output is taken from an intermediate stage of theamplifier to a high resolution counter 162, measuring the period ofresonant oscillation.

It is found that the frequency of oscillation varies with the populationof viable bacterium suspended in the medium. A decrease in the number ofviable bacteria is identified by a reduction of oscillation frequency.It is believed that this behaviour can be explained by the release ofadditional electrolyte into the medium which occurs on rupture of a cellwall. A decrease in the number of viable cells therefore results in areduction of the dielectric component but an increase in electrolyticconduction. The time constant therefore increases and the frequency ofresonance drops.

It should be understood that this invention has been described by way ofexamples only and a variety of modifications are possible withoutdeparting from the scope of the invention. Thus whilst reference hasbeen made throughout to test cells through which medium passescontinuously, apparatus according to this invention can function with astationary volume of medium in a test cell or indeed with electrodesdipped in a body of sample in the manner of a probe.

In terms of analysis, it has been found that with one particularbacterium--E Coli--investigation of the strength of the alpha dispersionreveals useful information concerning viability. Under othercircumstances and with different micro-organisms, alternative forms ofanalysis will be appropriate.

The invention in its various forms has particular application indetermining the proportion of bacteria in a population which are viable,but may have other applications in investigating the concentration ofbiological particles in a medium which may be a suspension, slurry orcolloid. In parts of the described methods, apparatus according to thisinvention is employed to determine the impedance of medium from whichbiological matter has been separated, in order to provide referencelevels. This usage may be extended to cases where the impedance (orconductivity) measurement of the medium is the primary concern.

We claim:
 1. Apparatus for investigating the condition of a suspensionof bacteria in a medium, comprising:a chamber comprising a conduitfilled with the medium in use; a pair of electrodes positioned tocontact said medium contained within the chamber; means for producing acontrolled continuous flow of said medium through said conduit betweensaid electrodes; voltage generator means electrically connected withsaid electrodes and serving to generate an alternating voltage ofselected frequency across said electrodes, thereby causing analternating current to flow in said medium therebetween; coil meansdisposed so that a time varying voltage is induced therein by atime-varying magnetic field associated with said alternating currentflow in the medium; means for measuring said induced voltage; andprocessor means, including means for controlling said voltage generatormeans to generate said alternating voltage at a plurality of frequenciesand for also controlling said measuring means to provide an outputcorresponding to said induced voltage at said frequencies for generatinga profile therefrom of measured induced voltage in the coil means as aplot thereof against a plurality of different selected frequencies ofthe alternating voltage applied to the electrode, said profile beingindicative of a condition of the bacteria suspended in said medium. 2.Apparatus according to claim 1, wherein:the processor means comprisesstorage means for storing at least one of said profiles.
 3. Apparatusaccording to claim 1, wherein:the conduit is cylindrical and the coilmeans comprises a helical coil having its axis orthogonal to the axis ofthe conduit, said electrodes being separated along said conduit. 4.Apparatus according to claim 1, wherein the electrode meanscomprises:the two electrodes are disposed to be one at each end of theconduit, both electrodes being apertured to permit flow of said mediumtherethrough.
 5. Apparatus for investigating a condition of a suspensionof bacteria in a medium, comprising:a chamber filled in use with acontrolled flow of the medium in a first direction; drive coil meanspositioned adjacent the chamber with an axis of the drive coil meansoriented to be orthogonal to said first direction; variable frequency,constant amplitude current drive means for causing an alternatingcurrent to flow at constant amplitude through the drive coil means, suchthat a time varying magnetic field associated with the drive coil meanscauses a corresponding alternating current to flow in the medium;detector coil means so disposed adjacent the chamber that a voltage isinduced therein by a time varying magnetic filed associated with saidalternating current flow in the medium, an axis of said detector coilmeans being oriented to be orthogonal to said first direction and tosaid drive coil means; means for measuring the voltage induced in thedetector coil means; and processor means, including means forcontrolling the variable frequency current drive means to generate saidvariable frequency constant amplitude current and also for controllingsaid measured induced voltage measuring means to provide an outputcorresponding to said induced voltage at said frequencies for generatingtherefrom a profile of measured induced voltage as a plot thereofagainst a plurality of different selected frequencies of said variablefrequency constant amplitude current, said profile being indicative of acondition of the bacteria suspended in said medium.
 6. Apparatusaccording to claim 5, wherein:the constant amplitude current drive meanscomprises a voltage generator means for applying an alternating voltageof variable frequency to said drive coil means, current sensing meansfor sensing a current flow in the drive coil means, and feedback meansfor varying the amplitude of said applied alternating voltage independence upon said sensed current flowing in the drive coil means. 7.Apparatus according to claim 5, wherein:said processor comprises storagemeans for storing profiles and comparator means for comparing profiles.